We have purified from Acanthamoeba castellanii a second myosin ATPase, Acanthamoeba myosin II, which induces syneresis when added to gels formed from F-actin and purified Acanthamoeba gelation factors. This myosin differs in many ways from the first myosin ATPase, Acanthamoeba myosin I, previously isolated from the same organism. Myosin II has a native molecular weight of about 350,000 derived from multiples of a heavy chain of about 170,000 daltons and two different light chains of about 17,500 and 17,000 daltons and is, therefore, a two-headed myosin. Its very low Mg2 ion -ATPase activity is stimulated only about two times by F-actin even in the presence of Acanthamoeba myosin I cofactor proteins. In contrast, myosin I is a single-headed enzyme of native molecular weight about 180,000 consisting of a heavy chain of 140,000 daltons and light chains of 16,000 and 14,000 daltons; Its Mg2 ions-ATPase activity is highly activated by F-actin in the presence of cofactor proteins. These, and other significant physical and enzymatic differences, indicate that Acanthamoeba myosin I and II are the first known examples of the occurrence of two different myosins in the same cell where they presumably have different functions. BIBLIOGRAPHIC REFERENCES: Maruta, H. and Korn, E.D.: Purification from Acanthamoeba castellanii of proteins that induce gelation and syneresis of F-actin. J. Biol. Chem. 252: 399-402, 1977. Korn, E. D.: Biochemistry of motility in Acanthamoeba castellanii. In Perry, S.V., Margreth, A. and Adelstein, R.S. (Eds.): Contractile Systems in Non-Muscle Tissues. Amsterdam-Oxford-New York, Elsevier/North Holland Biomedical Press, 1976, pp. 285-296.